Insulin-like growth factors (IGF)-I and II are potent mitogens for mammary tumors. We hypothesize that their growth modulatory effects are governed, in large part, by IGF-binding proteins (IGFBPs), a class of six recently cloned polypeptides which are synthesized and secreted by breast cancer cells. Using the hormone-responsive N-nitrosomethylurea (NMU)- induced rat mammary tumor, we plan to investigate the expression and regulation by hormones and polyamines of IGFs and IGFBPs in vivo. Because of the multihormonal dependency of the NMU tumor, we will be able to examine the individual and combined effects of different classes of hormones such as estradiol, prolactin and progesterone on the expression of these peptides. In situ hybridization and immunohistochemistry techniques will allow us to address the issues of heterogeneous expression by different subpopulations of breast cancer cells. Differences between neoplastic and normal mammary tissue in the same animals will also be examined. We plan to directly evaluate the actions of IGFBPs using transfection techniques and exogenous administration of IGFBPs in experimental and human breast cancer cells in culture. The following endpoints will be assessed in our transfected and/or IGFBP- treated cells: a) basal proliferative activity; b) sensitivity to the growth-stimulatory effects of exogenously added IGF-I and -II; c) estrogen sensitivity; d) sensitivity to antipolyamine therapy; and e) in vivo growth characteristics in nude mice. We will determine potential mechanisms regulating the release of IGFs from the IGFBPs to their target cells since they may be critically involved in IGF-stimulated tumor growth. These mechanisms may include phosphorylation and proteolytic processing of the IGFBPs and their interactions with the surface of stromal and epithelial cells. The influence of hormonal and polyamine manipulations on these processes will also be examined. Finally, the potential paracrine effects of the IGF system will be assessed by studying the growth modulatory effects of stromal cell-derived conditioned medium on target epithelial tumor cells. The dependence of the projected stimulatory response on components of the IGF system will be tested immunochemically using neutralizing antibodies or quantitative immunoprecipitation techniques.